Bottom Line:
We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability.A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish.Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein.

ABSTRACTA missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies.

Mentions:
The recombinant mutant α-DG(485–630)I591D, expressed as a fusion protein conjugated with six N-terminal histidine residues and the thioredoxin (Trx), was purified by affinity chromatography using a nickel nitrilotriacetate resin. After thrombin cleavage to separate α-DG(485–630)I591D from its fusion partner, the protein was submitted to a further affinity chromatography step to remove the fusion partner from the solution. A similar protocol was applied to the wild-type protein in order to compare the stability of the two proteins (Fig. 9). Any attempt made to further purify the I591D mutant was unsuccessful because of its high propensity to degradation. Figure 9 shows an SDS-PAGE, in which protein samples at different stages of the purification protocol were analyzed. The purified protein, compared to its wild-type counterpart, displays a faint band corresponding to the lower degraded band observed in the wild-type, while no signal corresponding to the full-length protein can be observed. At the present stage, due to this pronounced unstable behavior, it is actually impossible to collect significant amounts of the I591D variant to be employed for its biochemical characterization.

Mentions:
The recombinant mutant α-DG(485–630)I591D, expressed as a fusion protein conjugated with six N-terminal histidine residues and the thioredoxin (Trx), was purified by affinity chromatography using a nickel nitrilotriacetate resin. After thrombin cleavage to separate α-DG(485–630)I591D from its fusion partner, the protein was submitted to a further affinity chromatography step to remove the fusion partner from the solution. A similar protocol was applied to the wild-type protein in order to compare the stability of the two proteins (Fig. 9). Any attempt made to further purify the I591D mutant was unsuccessful because of its high propensity to degradation. Figure 9 shows an SDS-PAGE, in which protein samples at different stages of the purification protocol were analyzed. The purified protein, compared to its wild-type counterpart, displays a faint band corresponding to the lower degraded band observed in the wild-type, while no signal corresponding to the full-length protein can be observed. At the present stage, due to this pronounced unstable behavior, it is actually impossible to collect significant amounts of the I591D variant to be employed for its biochemical characterization.

Bottom Line:
We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability.A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish.Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein.

ABSTRACTA missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies.